Role of NADPH Oxidase-Derived ROS-Mediated IL-6/STAT3 and MAPK/NF-κB Signaling Pathways in Protective Effect of Corilagin against Acetaminophen-Induced Liver Injury in Mice

Simple Summary Acetaminophen overdose causes acute liver injury by excessive oxidative stress. The present study examines the mechanisms underlying the protective effect of corilagin against acetaminophen-induced liver injury. Our results showed that corilagin attenuates the liver injury through its anti-oxidant and anti-inflammatory properties. Corilagin may be a therapeutic agent for acute liver injury. Abstract Acetaminophen (APAP) overdose causes acute liver injury via oxidative stress, uncontrolled inflammatory response, and subsequent hepatocyte death. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a potent source of cellular reactive oxygen species (ROS) and may contribute to oxidative stress in many inflammatory processes. Corilagin, a component of Phyllanthus urinaria, possesses antioxidant, anti-inflammatory, and hepatoprotective effects. We evaluated the mechanisms underlying the protective effect of corilagin against acetaminophen-induced liver injury. Mice were intraperitoneally administrated 300 mg/kg APAP or equal volume of saline (control), with or without various concentrations of corilagin (0, 1, 5, or 10 mg/kg) administered after 30 min. All animals were sacrificed 16 h after APAP administration, and serum and liver tissue assays including histology, immunohistochemistry, and Western blot assay were performed. Corilagin post-treatment significantly attenuated APAP-induced liver injury (p < 0.005), inflammatory cell infiltration, hepatic proinflammatory cytokine levels, and hepatic oxidative stress. Furthermore, corilagin attenuated the protein levels of NOX1, NOX2, signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappa B (NF-κB) in APAP-induced liver injury. These results indicated that the antioxidant, anti-inflammatory, and protective effects of corilagin in APAP-induced liver injury might involve the regulation of interleukin (IL)-6/STAT3 and mitogen-activated protein kinase (MAPK)/NF-κB signaling pathways through NOX-derived ROS.


Introduction
Acetaminophen, N-acetyl-p-aminophenol (APAP), is commonly used for antipyretics and analgesics globally [1]. APAP is considered safe at proper therapeutic doses; however, accidental or unintentional overdose may lead to acute liver injury [2,3]. APAP is metabolized to its inactive glucuronide and sulfate conjugates in liver [4]. Only a small amount of APAP is oxidized to the metabolite N-acetyl-p-benzoquinone imine (NAPQI), which is inactivated to form non-toxic compounds [5,6]. Excessive NAPQI formation can result in oxidative stress and uncontrolled inflammatory response [3,7,8].

Effects of Corilagin on Hepatic Accumulation of Neutrophils and Macrophages in APAP-Induced Hepatic Injury
Immunohistochemistry of liver tissue sections using an antibody to Ly6G, a granulocyte-specific marker, revealed overt neutrophil infiltration of the necrotic areas in the APAP group ( Figure 2). The groups treated with corilagin following APAP administration exhibited significantly decreased neutrophil infiltration in liver parenchyma. Figure 1. The effects of corilagin on serum ALT levels (A), AST levels (B), and histopathological changes (C) in acetaminophen-induced hepatic injury. Mice were injected with APAP (300 mg/kg) or equal volume of saline (control), and treated with corilagin (0, 1, 5, or 10 g/kg) after 30 min. Each value represents mean ± SEM; n = 6 for each group. ## p < 0.01, ### p < 0.005 vs. control group; ** p < 0.01, *** p < 0.005 vs. APAP group (50×). Histological examination demonstrated severe centrilobular hepatic necrosis in the APAP group. Clearly, the treatment with 5 and 10 mg/kg corilagin attenuated the pathological changes ( Figure 1C).

Effects of Corilagin on Hepatic Accumulation of Neutrophils and Macrophages in APAP-Induced Hepatic Injury
Immunohistochemistry of liver tissue sections using an antibody to Ly6G, a granulocytespecific marker, revealed overt neutrophil infiltration of the necrotic areas in the APAP group ( Figure 2). The groups treated with corilagin following APAP administration exhibited significantly decreased neutrophil infiltration in liver parenchyma. Animals were injected with APAP (300 mg/kg) or equal volume of saline (control), and treated with corilagin (0, 1, 5, or 10 g/kg) (200×). Each value represents mean ± SEM; n = 6 for each group. ### p < 0.005 vs. control group; *** p < 0.005 vs. APAP group.
To evaluate macrophage infiltration following APAP-induced injury, the liver tissue sections were immunohistochemically stained using an antibody to Mac-2. The APAP group exhibited increased macrophage infiltration of the necrotic areas, whereas the groups treated with corilagin following APAP administration exhibited significantly decreased macrophage infiltration in liver parenchyma ( Figure 3). Animals were injected with APAP (300 mg/kg) or equal volume of saline (control), and treated with corilagin (0, 1, 5, or 10 g/kg) (200×). Each value represents mean ± SEM; n = 6 for each group. ### p < 0.005 vs. control group; *** p < 0.005 vs. APAP group.
To evaluate macrophage infiltration following APAP-induced injury, the liver tissue sections were immunohistochemically stained using an antibody to Mac-2. The APAP group exhibited increased macrophage infiltration of the necrotic areas, whereas the groups treated with corilagin following APAP administration exhibited significantly decreased macrophage infiltration in liver parenchyma ( Figure 3).

Effects of Corilagin on Hepatic IL-6 and TNF-α Levels
Hepatic IL-6 and TNF-α levels, which were increased in the APAP group, did not differ between the control and 10 mg/kg corilagin alone groups ( Figure 4A). Importantly, treatment with 5 mg/kg corilagin significantly reduced hepatic IL-6 levels (p < 0.01). Moreover, IL-6 and TNF-α were lower in the group treated with 10 mg/kg corilagin following APAP administration ( Figure 4B).

Corilagin on Hepatic MDA and GSH Levels and MPO Activity
In Figure 5, MDA, a lipid peroxidation marker, which was markedly increased in the APAP group, was reduced after the treatment with 5 and 10 mg/kg corilagin. Moreover, hepatic levels of GSH, a cellular antioxidant marker, which was significantly lower in the APAP group, was significantly higher after the treatment with 1, 5, and 10 mg/kg corilagin. Finally, MPO activity, used as a marker for neutrophil infiltration and oxidant levels, which was increased in the APAP group, was decreased after the treatment with corilagin. To evaluate macrophage infiltration following APAP-induced injury, the liver tissue sections were immunohistochemically stained using an antibody to Mac-2. The APAP group exhibited increased macrophage infiltration of the necrotic areas, whereas the groups treated with corilagin following APAP administration exhibited significantly decreased macrophage infiltration in liver parenchyma ( Figure 3).  Animals were injected with APAP (300 mg/kg) or equal volume of saline (control), and treated with corilagin (0, 1, 5, or 10 g/kg) (200×). Each value represents mean ± SEM; n = 6 for each group. ## p < 0.01, ### p < 0.005 vs. control group; *** p < 0.005 vs. APAP group.

Corilagin on Hepatic MDA and GSH Levels and MPO Activity
In Figure 5, MDA, a lipid peroxidation marker, which was markedly increased in the APAP group, was reduced after the treatment with 5 and 10 mg/kg corilagin. Moreover, hepatic levels of GSH, a cellular antioxidant marker, which was significantly lower in the APAP group, was significantly higher after the treatment with 1, 5, and 10 mg/kg corilagin. Finally, MPO activity, used as a marker for neutrophil infiltration and oxidant levels, which was increased in the APAP group, was decreased after the treatment with corilagin. . The effects of corilagin on IL-6 (A) and TNF-α (B) concentrations in acetaminophen-induced hepatic injury. Animals were intraperitoneally injected with APAP (300 mg/kg) or equal volume of saline (control), and administrated with various concentrations of corilagin (0, 1, 5, or 10 g/kg) after 30 min. # p < 0.05, ### p < 0.005 vs. control group; * p < 0.05, ** p < 0.01, *** p < 0.005 vs. APAP group.

Effects of Corilagin on IL-6 and STAT3 Concentrations
We measured hepatic IL-6 and STAT3 concentrations using Western blotting. Hepatic IL-6 and STAT3 levels, which were not different between the control and 10 mg/kg corilagin alone groups, were significantly higher in the APAP group ( Figure 6C,D). Additionally, administration with corilagin following APAP administration significantly reduced hepatic IL-6 and STAT3 levels (p < 0.005 for both).

Discussion
Our analyses revealed that corilagin post-treatment reduced APAP-induced hepatic injury, inflammation, and oxidation. Moreover, corilagin post-treatment significantly reduced hepatic NOX1, NOX2, IL-6, STAT3, phospho-ERK, phospho-JNK, and NF-κB levels. Altogether, these results show the protection of corilagin against APAP-induced hepatic injury and suggest that its antioxidant and anti-inflammatory effect occurs through NOX-derived ROS-mediated regulation of the IL-6/STAT3 and MAPK/NF-κB.
Innate immune system is important for APAP-induced hepatic injury [26][27][28]. Hepatocyte apoptosis or necrosis activate Kupffer cells through recognition by TLR [29][30][31]. Hepatic recruitment of monocytes and neutrophils secrete proinflammatory cytokines that contribute to an extensive inflammatory response and the subsequent severe liver damage [32][33][34][35]. Consistent with our previous work, we herein demonstrate that corilagin post-treatment ameliorates APAP-induced hepatic injury and reduces inflammatory cell infiltration and proinflammatory cytokine levels, confirming that corilagin exhibits protective effects for APAP-induced hepatic injury.
Progressive oxidative stress is characterized for APAP-induced hepatic injury [36,37]. Oxidative stress is a function of ROS overproduction in the setting of relative deficiency in antioxidant defenses [38,39]. Imbalanced ROS generation and consequent free radical production may result in injury [40]. Malondialdehyde is the main final product of lipid peroxidation in cells, and malondialdehyde levels frequently serve as an indicator of oxidative stress [41]. Myeloperoxidase represents the degree of neutrophil infiltration, and myeloperoxidase levels are a common biomarker of oxidative damage [42]. A recent study demonstrated that corilagin reduced oxidative stress in APAP-induced hepatic injury [43]. In agreement, we found that corilagin post-treatment led to reductions in malondialdehyde and myeloperoxidase levels, indicating reduced ROS production. Moreover, we found that corilagin post-treatment led to increased glutathione levels, indicating enhanced antioxidant capacity. Altogether, the results show that corilagin exerts protection against APAP-induced liver injury through antioxidant mechanisms.
A previous study demonstrated that excess ROS production mediated through NOX activation played a vital role in neutrophil-mediated inflammatory response and liver injury in a hemorrhagic shock model [44]. A recent study reported that NOX1-derived ROS played a critical role in liver disease [45]. Moreover, NOX2, primarily expressed in Kupffer cells, macrophages, and neutrophils, plays an important role in inflammation through ROS release in many inflammatory conditions [46]. Furthermore, we recently reported that corilagin post-treatment attenuated lipopolysaccharide-induced lung injury through the inhibition of NOX2 pathway [24]. We found that the marked increase in hepatic NOX1 and NOX2 levels was attenuated with corilagin post-treatment, suggesting that the antioxidant effect of corilagin was, at least partially, through the downregulation of NOX1 and NOX2 levels in liver.
STAT3, a transcription factor activated by several proinflammatory cytokines, such as IL-6, regulates diverse cellular functions, such as inflammation, survival, differentiation, and proliferation [47]. STAT3 signaling is associated with liver injury, fibrosis, inflammatory response, and oncogenesis [48]. Previous studies have shown the significant role of STAT3 in hepatic injury [49]. A recent study demonstrated that treatment with a STAT3 inhibitor attenuated acute liver injury by regulating macrophages and reducing proinflammatory cytokine levels [50]. Corilagin post-treatment reduced IL-6 and STAT3 levels, suggesting that the anti-inflammatory effect of corilagin might occur via the regulation of IL-6/STAT3. MAPK family members, including ERK and JNK, play essential roles in differentiation, inflammation, and oxidative stress [51]. MAPK activation can lead to the induction of NF-κB pathway and subsequent increases in proinflammatory cytokine secretion and augmentation of inflammation [52]. Consistent with our previous work, we found that APAP challenge led to significant increases in phospho-JNK, phospho-ERK, and NF-κB, which were significantly reduced with corilagin post-treatment, suggesting that the antiinflammation of corilagin was through MAPK signaling pathway.

Conclusions
In conclusion, corilagin was beneficial for APAP-induced hepatic injury through its antioxidation and anti-inflammation. The underlying mechanisms might involve the regulation of IL-6/STAT3 and MAPK/NF-κB mediated with NOX-derived ROS.